Studies of Rabbit CD5 The variety of combining sites generated within individual clones from appendix follicles of young rabbits suggests that clonal expansion and selection, known to require gut flora, may not be driven by specific antigens but rather through indirect effects of microbial components (superantigens). In addition, there may be survival signals supplied by interactions between B-cell receptor framework regions and endogenous superantigens such as CD5. Although the function of CD5 on B cells is unknown, our studies in the rabbit, suggested that CD5 interaction with VH framework regions of surface immunoglobulins may contribute to survival and expansion of B cells. For further studies of the interaction of CD5 and VH-regions, a fragment of CD5 protein containing the three extracellular domains has been cloned and expressed as recombinant protein. Purified CD5 protein was also used to produce polyclonal goat anti-rabbit CD5. In addition we generated anti-peptide mAbs specific for each extracellular domain of CD5. These reagents are being used to continue a detailed analysis of the development and selection of rabbit appendix B lymphocytes (1). Although only a small proportion of mouse and human B cells are CD5+, most adult rabbit B cells express CD5. However, CD5 was not detectable on the majority of B cells in neonatal appendix 1 and 3 days after birth. Cell trafficking studies demonstrated that CD5+ and CD5- CD62L+ B cells from bone marrow migrated into appendix. There, CD5+ B cells were preferentially expanded and predominated by ~2 weeks of age. In mutant ali/ali rabbits, VHa2+ B cells develop through gene conversion-like alteration of rearranged VH genes upstream of deleted VH1a2. Correlated appearance of individual CD5+ germinal centers and VHa2+ B-cells in mutants' appendix suggest that CD5 binding positively selects cells with a2+ framework regions that bind CD5. Following negative and positive selection, cells with diversified rearranged heavy- and light-chain sequences exit appendix, migrate to peripheral tissues and constitute the preimmune repertoire of CD5+ B cells that encounter foreign antigens. (Pospisil, R. et al. CD5+ B cells are preferentially expanded in rabbit appendix: the role of CD5 in B cell development and selection, submitted 2005). Studies of Rabbit Activation Induced Deaminase Studies in mouse, human and chicken suggest that activation-induced deaminase (AID) is involved in the three known processes leading to antibody diversification: somatic hypermutation, gene conversion, and class switch recombination. Developing rabbit appendix provides a particularly good site for studying all three of these B-cell maturation events. We extended knowledge about AID to a mammalian species that uses gene conversion to diversify rearranged immunoglobulin genes by cloning and sequencing rabbit AID, isolating AID protein from rabbit appendix-cell nuclear and cytoplasmic extracts (identity confirmed by mass spectrometry). We produced anti-AID antibody that identified AID protein in cells by immuno-histochemical and -fluorescent staining techniques and described co-localization of AID and other molecules important for Ab diversification. Although much work remains to understand fully the mechanism of action of AID and its association with other cellular components, the rabbit system now offers a particularly useful model for future studies of these dynamics (Yang, G. et al, Activation-induced Deaminase: Cloning, localization, and protein extraction from young VH-mutant rabbit appendix. Submitted, 2005) Recruitment of Blood-Borne B-cells to Neonatal Rabbit Appendix Young rabbit appendix is a homologue of chicken bursa of Fabricius; both are crucial sites for preimmune B-cell repertoire development. These are primary lymphoid organs where the B cell antibody repertoire develops in germinal centers mainly by a gene conversion-like process. Although B cell Ig-gene rearrangements occur in sites such as bone marrow of young rabbits, immature IgM+ B cells undergo further Ig-repertoire diversification in appendix and other gut associated lymphoid tissues. We have now characterized some of the molecules involved in the multi-step recruitment of blood-borne B cells into neonatal rabbit appendix. Expression of peripheral lymph node addressin (PNAd) on appendix high endothelial venules (HEV), and of its counter-receptor, CD62L, on B cells in peripheral blood and in close association with PNAd-positive HEV suggests their role in tethering. Sialyl-Lewis-x, known to be involved in tethering of pre-bursal cells on chicken bursal vasculature, was also found on appendix B cells. The interaction of chemokine receptor CCR7 on peripheral blood B cells and one of its ligands, CCL21, on appendix HEV, could cause activation of integrins such as LFA-1 and alpha 4/beta-1 and lead to firm adhesion of B cells to HEV. We found these integrins expressed on peripheral blood B cells. Simultaneous down-regulation of CCR7 and up-regulation of CXCR5 on appendix B cells compared to peripheral blood suggests a role for CXCR5 in B-cell recruitment into follicles during appendix development. The proportions of appendix B cells expressing CD62L, sialyl-Lewis-x and alpha-4/beta-1 declined between day 3 and 4 weeks after birth while percentages of Lewis-x+ appendix B cells increased. These changes correlate with the stage of repertoire diversification by gene conversion in both rabbits and chickens (1). We found that appendix regulates precursor lymphocyte recruitment for further development by modulating the sites of extravasation. The total area of peripheral node addressin-positive (PNAd+) high endothelial venules (HEVs) increased from 1-day to 1-week after birth, remained constant up to 2-weeks and declined to a low and persistent amount by 3-weeks. In normal 1-week and manipulated 5-week-appendix where growth of follicles was retarded, PNAd+ HEVs were present in the basolateral sides of B-cell follicles whereas, in normal 5-wk-appendix these were restricted to T-cell areas. The PNAd was an ~120 kDa O-sialoglycoprotein expressed on the lumenal surface of HEVs. The proportions of CD62L+ B cells in appendix declined from ~40% at 3-days to 2-3% at 4-weeks. In lymphocyte transfer experiments, CD62L+ B cells were preferentially recruited compared with CD62L- B cells, anti-PNAd antibody blocked migration of B cells by ~50%, and 100 times more B cells were recruited in 1-week compared to 6-week appendix. Thus, a unique spatiotemporal expression pattern of PNAd+ HEVs is associated with development of B-cell follicles. This regulates migration of blood-borne B-lymphocytes into developing appendix by interacting with L-selectin (CD62L) (Sinha, R., et al. Regulated expression of peripheral node addressin-positive high endothelial venules controls seeding of B lymphocytes into developing neonatal rabbit appendix, Submitted 2005). The Kit activating Mutation in Mast Cells, and Oligoclonal B-Lymphocytes of Mastocytosis Patients To explore the relationship of the occurrence of a somatically acquired activating Kit D816V mutation to the heterotypic clusters of mast cells and lymphocytes in bone marrow of mastocytosis patients, laser capture microdissected mast cells, B cells and T cells, from both lesional and non lesional areas of bone marrow biopsy tissues from patients with mastocytosis,were examined for the kit mutation. We found mast cells and lymphocytes within focal aggregates in the bone marrow of those with mastocytosis are more frequently positive for the kit mutation and that the B cell population in lesional areas of mastocytosis as well as control B cells in normal appendix represented oligoclonal populations. Clonal proliferation is unlikely to be the basis of clustering of B cells in lesional areas of patient bone marrow (3).